Project Summary Opioid use among pregnant women is a growing public health concern in the United States. Infants exposed to opioids in utero are at high risk of exhibiting Neonatal Opioid Withdrawal Syndrome (NOWS), a combination of physical withdrawal symptoms including high pitched crying, sleeplessness, irritability, gastrointestinal distress, and in the worst cases, seizures. The complexity of this syndrome is amplified by a variety of clinical factors such as duration of maternal opioid exposure, maternal polypharmacy, environment, and genetics. The complexity of in utero opioid exposure and NOWS make it very difficult to investigate underlying molecular mechanisms that could ultimately inform early diagnosis and treatment. Therefore, we have developed a much needed mouse model of 3- trimester opioid exposure and withdrawal. Based on preliminary evidence, we hypothesize that microglia activation and immune mediators contribute to the severity of NOWS and a common SNP in the ?-opioid receptor modulates these effects. Using morphine as a prototypical opioid, we will exploit our model to test these hypotheses. First we will fully characterize the role of the innate immune system in NOWS and determine if regulation of neuroinflammation has therapeutic potential by pharmacological treatment with the anti- inflammatory drug, ibudilast. Second, as recent clinical findings suggest that genomic variation in the gene that encodes the ?-opioid receptor (Oprm1 A118G) may influence NOWS severity, we will use our mouse line that contains the equivalent Oprm1 A118G SNP to determine the impact of Oprm1 genetics on microglia activation and immune mediators. Using our model of 3-trimester opioid exposure and withdrawal we will use TRAP-Seq, a new technology for retrieving mRNAs within the ribosomal complex that are actively being translated, in our case in cells expressing m-opioid receptors, to interrogate the changing transcriptome following opioid exposure and withdrawal. This project is likely to have a sustained and powerful impact on the field because we will address mechanisms through which perinatal exposure to opiates results in NOWS, and how genetics and immune response contribute.